CN114235933A

CN114235933A - A kind of dyestuff of colored polyacrylamide gel and preparation method thereof

Info

Publication number: CN114235933A
Application number: CN202111576920.4A
Authority: CN
Inventors: 姜自运; 宋斌斌; 张佳佳
Original assignee: Anhui Haotuo Biotechnology Co ltd
Current assignee: Anhui Haotuo Biotechnology Co ltd
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-03-25

Abstract

The invention discloses a dye for colored polyacrylamide gel and a preparation method thereof, belonging to the technical field of protein separation in the field of biological electrophoresis. In the present invention, under alkaline conditions, sodium dithionate emits electrons, and the dye molecules receive electrons and are reduced to soluble sodium salts, which solves the problem that carbonyl dyes cannot be directly dissolved in water, and the dyes have bright colors and rich varieties. The adjustable cavity makes the preparation process of polyacrylamide gel more convenient and safe. The color stacking gel is easy to prepare, and the dye properties are stable. It will not migrate with electrophoresis and will not affect the normal migration of proteins, making polyacrylamide gel electrophoresis. The spotting of the sample is more convenient, and the observation of the sample hole is high.

Description

Dye of colored polyacrylamide gel and preparation method thereof

Technical Field

The invention belongs to the technical field of protein separation in the field of biological electrophoresis, and particularly relates to a dye of colored polyacrylamide gel and a preparation method thereof.

Background

Polyacrylamide gel electrophoresis (also called page) is an electrophoretic technique using polyacrylamide gel as a supporting medium, and is commonly used for the separation of protein samples. It is divided into two forms of Native-PAGE (Native-PAGE) and denaturing-polyacrylamide-gel-electrophoresis (SDS-PAGE). Mainly comprises a three-dimensional network structure formed by acrylamide monomer and cross-linking agent methylene bisacrylamide under the catalysis of ammonium persulfate. In SDS-PAGE, SDS has a large negative charge, and forms SDS-protein complex in combination with protein, and the negative charge of the complex is far more than that of the protein, so that the difference between the charges of the proteins is masked. The complex of SDS-protein now takes the shape of an oval rod, which is only related to the molecular weight of the protein subunits, so that the proteins can be separated only according to the molecular weight of the protein subunits upon electrophoresis. SDS-PAGE gel commonly used in laboratories is a discontinuous polyacrylamide gel system and is divided into concentrated gel and separation gel, wherein the concentrated gel is positioned at the upper layer of a vertical electrophoresis system during electrophoresis and has a concentration effect, the separation gel is positioned at the lower layer, and protein samples can be separated according to molecular weight after entering the separation gel, so that the SDS-PAGE gel is the most effective means for separating and detecting samples used in laboratories at present.

However, in the actual gel preparation and electrophoresis process, the polyacrylamide gel has some disadvantages, especially because the gel is colorless and transparent, the sample loading hole is not clear, and especially for novices, the situation that the sample loading hole is difficult to distinguish and wastes samples easily occurs; secondly, the gel has a complex formula, and various solutions with different contents need to be added one by one, and in the process, experimenters can frequently contact with toxic reagents such as acrylamide, TEMED and the like, so that the physical health of the experimenters is seriously damaged.

Disclosure of Invention

The invention aims to: the dye for the colored polyacrylamide gel and the preparation method thereof are provided in order to solve the problems that the polyacrylamide gel is colorless and transparent, sample loading holes are not clear, and sample loading holes are difficult to distinguish and waste.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a dye of colored polyacrylamide gel specifically comprises the following steps:

s101, performing the following steps of 1: respectively adding original dye molecules and water in a ratio of 18-22, and uniformly mixing;

s102, adding 25-35mL/L of 34-38% sodium hydroxide solution, uniformly stirring, and adjusting the pH value of the solution to be alkaline;

s103, heating the solution prepared in the S102 to 55-65 ℃, and keeping the temperature at a constant temperature for reservation;

s104, slowly adding sodium hydrosulfite to make the final concentration be 4.8-5.2 g/L;

s105, uniformly stirring the solution prepared in the step S104, and carrying out heat preservation reaction for 10min to obtain a gel dye mother solution;

s106, adding a proper amount of gel dye mother liquor in the process of preparing the polyacrylamide gel to enable the polyacrylamide gel to have a bright color.

As a further description of the above technical solution:

the original dye molecules are one or more of hydroxyl dyes.

As a further description of the above technical solution:

the original dye molecular structure contains no water-soluble group.

As a further description of the above technical solution:

the carbonyl dye is any one of anthraquinone dye or thioindigo dye.

As a further description of the above technical solution:

the chemical reaction equation of the gel dye preparation step is as follows:

under alkaline conditions, sodium dithionate emits electrons, dye molecules receive the electrons and are reduced into soluble sodium salts, and gel dye mother liquor which can be used for polyacrylamide gel is obtained.

As a further description of the above technical solution:

dye of colored polyacrylamide gel the steps for preparing colored polyacrylamide gel are as follows:

s201, preparation of separation gel: adding 30% of Acr-Bis, 1.5M Tris-HCl (pH8.8), 10% of SDS and deionized water according to the concentration of the dyed gel, uniformly mixing, then adding 10% of ammonium persulfate and TEMED according to the formula amount, quickly filling the solution between the assembled vertical electrophoresis glass plates after uniform mixing, enabling the upper edge of the separation gel to be about 3cm away from the upper edge of the glass plates, adding a small amount of water into the separation gel, standing until the separation gel is polymerized, and then absorbing the water layer by using filter paper;

s202, preparation of concentrated glue: adding 30% of Acr-Bis, 1.0M Tris-HCl (pH6.8), 10% of SDS deionized water and a proper amount of gel dye mother liquor into a beaker according to the proportion, uniformly mixing, wherein the final concentration of the gel dye mother liquor is generally 0.05-1.0mg/mL, then adding 10% of ammonium persulfate and TEMED according to the formula amount, uniformly mixing, pouring the solution above a separation gel to be full, slowly inserting into a comb, standing until the solution is solidified, and carrying out electrophoresis.

As a further description of the above technical solution:

the prepared colored polyacrylamide gel is used for separating protein samples, and specifically comprises the following steps:

s301, mixing concentrated gel buffer solution with gel dye to prepare color polyacrylamide gel premix;

s302, when glue is prepared, uniformly mixing a separating glue buffer solution and a separating glue solution, and adding gel to promote gelation to obtain direct glue filling;

s303, when preparing the color concentrated glue, mixing the concentrated glue buffer solution and the concentrated glue solution, adding the mixed solution into the gel coagulant to obtain the concentrated glue gel with different colors without adding extra dye.

As a further description of the above technical solution:

the premixed liquid comprises a separation gel buffer solution, a separation gel solution, a concentrated gel buffer solution, a concentrated gel solution and a gel coagulant, and is prepared from the following components:

color concentrated gel buffer: 1.0M Tris-HCl (pH6.8), TEMED, dH2O, gel dye;

separating gel buffer solution: 1.5M Tris-HCl (pH8.8), TEMED, dH 2O;

separating the gum solution: 30% Acr-Bis, dH 2O;

concentrating the gum solution: 30% Acr-Bis, dH 2O;

gel setting accelerator: 10% ammonium persulfate.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

in the invention, sodium dithionate emits electrons under alkaline conditions, dye molecules receive the electrons and are reduced into soluble sodium salt, the problem that carbonyl dye can not be directly dissolved in water is solved, a method for preparing dye capable of coloring polyacrylamide gel is provided, the dye is bright in color and rich in variety, the dyeing effect can be achieved through anthraquinone and thioindigo dyes with various colors, a dye color adjustable cavity enables the preparation process of the polyacrylamide gel to be more convenient and safer, color concentrated gel is easy to prepare, the dye is stable in property and cannot migrate along with electrophoresis, the normal migration of protein cannot be influenced, the sample application of polyacrylamide gel electrophoresis is more convenient, the observability of a sample loading hole is high, whether a sample is accurately added into the sample loading hole or not can be judged, whether the sample loading hole is damaged or distorted or not can be judged, and when multiple groups of electrophoresis are required, different gels have different concentrated gel colors and can be used for distinguishing different electrophoreses of different samples.

Drawings

FIG. 1 is a polyacrylamide gel containing different concentration gradients of green dye prepared in example 1;

FIG. 2 is a graph showing the effect of loading green polyacrylamide gel having a dye concentration of 0.3mg/mL in an electrophoresis tank in example 1;

FIG. 3 is a picture of example 1 after electrophoresis on a green polyacrylamide gel having a dye concentration of 0.3 mg/mL;

FIG. 4 is a photograph of a gel of a protein band after electrophoresis on a green polyacrylamide gel having a dye concentration of 0.3mg/mL in example 1 and staining with Coomassie Brilliant blue;

FIG. 5 is a red polyacrylamide gel prepared in example 2;

FIG. 6 is a photograph of the red polyacrylamide gel prepared in example 2 after electrophoresis;

FIG. 7 is a purple polyacrylamide gel prepared in example 3;

FIG. 8 is a schematic representation of the purple polyacrylamide gel prepared in example 3 after electrophoresis;

FIG. 9 shows a concentrated gel buffer of colored polyacrylamide gel prepared in example 4;

FIG. 10 shows a conventional polyacrylamide gel prepared in the comparative example.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a preparation method of a dye of colored polyacrylamide gel specifically comprises the following steps:

The original dye molecules are one or more of hydroxyl dyes, the molecular structure of the original dye does not contain water-soluble groups, and the carbonyl dye is any one of anthraquinone dyes or thioindigo dyes;

the chemical reaction equation of the gel dye preparation step is as follows:

under the alkaline condition, sodium dithionate emits electrons, dye molecules receive the electrons and are reduced into soluble sodium salt, and gel dye mother liquor which can be used for polyacrylamide gel is obtained;

color concentrated gel buffer: 1.0M Tris-HCl (pH6.8), TEMED, dH2O, gel dye;

separating gel buffer solution: 1.5M Tris-HCl (pH8.8), TEMED, dH 2O;

separating the gum solution: 30% Acr-Bis, dH 2O;

concentrating the gum solution: 30% Acr-Bis, dH 2O;

gel setting accelerator: 10% ammonium persulfate.

Example 1

Preparation of Green gel dyes

The original dye molecules of the Green gel dye used in this example are one or more of Green dyes in anthraquinone dyes, specifically Vat Green 1;

adding 10g of anthraquinone dye Vat Green 1 into 200mL of deionized water, and uniformly mixing;

adding 6mL of 36% sodium hydroxide solution, stirring uniformly, and adjusting the pH value to be alkaline;

heating the solution to 60 ℃ and preserving heat;

slowly adding 1g of sodium dithionite to a final concentration of about 5 g/L;

stirring uniformly, and keeping the temperature to react for 10-15min to obtain green gel dye mother liquor.

Preparation of green polyacrylamide gel

Preparation of separation gel

Adding required reagents in sequence according to the following table, mixing uniformly, pouring into a glass plate assembled in advance, separating the liquid level of the gel from the upper edge of the glass by about 3cm, adding a small amount of deionized water to press the gel to make the liquid level, and after the gel is polymerized, absorbing the water layer by using filter paper.

Preparation of green concentrated glue

Adding the required reagents such as deionized water, 30% Acr-Bis, 1.0M Tris-HCl (pH6.8), 10% SDS and the like in sequence according to the following table, mixing uniformly, adding the green gel dyes with different concentration gradients respectively, mixing uniformly, adding 10% ammonium persulfate and TEMED according to the formula amount respectively, mixing uniformly, pouring the gel solutions with different colors above the separation gel respectively, slowly inserting a comb, standing to polymerize the gel, and thus obtaining the polyacrylamide gel of the green concentrated gel with different color gradients.

After the green polyacrylamide gels with different concentration gradients are solidified, as shown in figure 1, the effect of loading the polyacrylamide gel with the dye concentration of 0.3mg/mL into an electrophoresis tank is shown in figure 2, the concentrated gel is bright green, and the sample loading holes can be clearly observed.

Taking polyacrylamide gel with the concentration of concentrated gel dye of 0.3mg/mL to perform gel electrophoresis under the electrophoresis condition of Tris-Glycine buffer solution electrophoresis at 150V for 1h as a gel image after electrophoresis shown in FIG. 3; the green gel dye carried by the concentrated gel does not migrate along with electrophoresis and still remains in the original place of the gel;

as shown in FIG. 4, when the gel was stained with Coomassie brilliant blue fast staining solution and photographed by a gel imager, the protein bands in FIG. 4 were normal, and no band distortion or protein mobility change occurred, indicating that the gel dye did not affect the normal electrophoretic migration of the protein.

The above results all prove that the gel dye used in example 1 of the present invention can make polyacrylamide gel electrophoresis simpler and more convenient, and does not have any adverse effect.

Example 2

Preparation of red polyacrylamide gel

Preparation of Red gel dyes

The original dye molecules of the Red gel dye used in this example are one or more of Red dyes in thioindigo dyes, specifically Vat Red 1;

adding 10g of thioindigo dye Vat Red 1 into 200mL of deionized water, and uniformly mixing;

heating the solution to 60 ℃ and preserving heat;

slowly adding 1g of sodium dithionite to a final concentration of about 5 g/L;

stirring uniformly, and reacting for 10-15min under heat preservation to obtain red gel dye mother liquor.

Preparation of red polyacrylamide gel

The procedure for preparing the red polyacrylamide gel of this example was the same as that of example 1, wherein the final concentration of the red dye in the concentrated gel was 0.3 mg/mL.

Example 3

Preparation of purple polyacrylamide gel

Preparation of purple gel dyes

The original dye molecules of the Violet gel dye used in this example are one or more of the Violet dyes in isoviolanthrone anthraquinone dyes, specifically Vat Violet 1;

adding 10g of isoviolanthrone anthraquinone dye Vat Violet 1 into 200mL of deionized water, and uniformly mixing;

heating the solution to 60 ℃ and preserving heat;

slowly adding 1g of sodium dithionite to a final concentration of about 5 g/L;

stirring uniformly, and keeping the temperature to react for 10-15min to obtain the purple gel dye mother liquor.

Preparation of purple polyacrylamide gel

The procedure for the preparation of the purple polyacrylamide gel of this example was the same as that of example 1, wherein the final concentration of the purple dye in the concentrated gel was 0.15 mg/mL.

Example 4

Colored polyacrylamide gel premix

In order to make the preparation process of the polyacrylamide gel more convenient, the calculation and the addition of the gel dye are not required to be independently added during each gel preparation, and concentrated gel buffer solution with the gel dye can be mixed to prepare the colored polyacrylamide gel premix solution, so that the gel is convenient to prepare. The premixed liquid comprises a separation gel buffer solution, a separation gel solution, a concentrated gel buffer solution, a concentrated gel solution and a gel coagulant, and is prepared from the following components:

color concentrated gel buffer: 1.0M Tris-HCl (pH6.8), TEMED, dH2O, gel dye separation gel buffer: 1.5M Tris-HCl (pH8.8), TEMED, dH2O

Separating the gum solution: 30% Acr-Bis, dH2O

Concentrating the gum solution: 30% Acr-Bis, dH2O

Gel setting accelerator: 10% ammonium persulfate

When the gel is prepared, the direct gel filling can be obtained only by uniformly mixing the separating gel buffer solution and the separating gel solution and adding the gel to promote the gelation; similarly, when the color concentrated glue is prepared, the concentrated glue buffer solution and the concentrated glue solution are mixed and added into the gel coagulant to obtain the concentrated glue gel with different colors without adding extra dye, thereby saving the steps. The color concentrated gel buffer prepared in this example is shown in FIG. 9, and has various colors and can be taken and used at any time;

comparative example: ordinary polyacrylamide gel

The gel preparation method is the same as that of the separation gel and the concentrated gel in example 1, but gel dye is not added, and after the gel is solidified, as shown in figure 10, the common polyacrylamide gel is completely transparent, so that the separation gel and the concentrated gel cannot be clearly distinguished, and the sample loading hole cannot be distinguished at a glance.

The embodiment shows that the colored polyacrylamide gel prepared by the method can realize higher sample treatment effect, the colored concentrated gel is easy to prepare, the dye property is stable, and the migration of the dye along with electrophoresis is avoided, so that the normal migration of protein is not influenced.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. a preparation method of the dye of colored polyacrylamide gel, is characterized in that, specifically comprises the following steps:

S101. Add original dye molecules and water in a ratio of 1:18-22, and mix them evenly;

S102, adding 25-35mL/L of 34-38% sodium hydroxide solution, stirring evenly, and adjusting the pH value of the solution to be alkaline;

S103, heating the solution prepared in S102 to 55-65° C. and keeping it at a constant temperature for standby;

S104, slowly adding sodium dithionite, so that the final concentration is 4.8-5.2g/L;

S105, the solution prepared in S104 is stirred evenly, and the gel dye mother liquor can be obtained by holding the reaction for 10 min;

S106, in the process of preparing the polyacrylamide gel, add an appropriate amount of gel dye mother liquor to make it have bright colors.

2 . The method for preparing a dye for colored polyacrylamide gel according to claim 1 , wherein the original dye molecule is one or more of hydroxyl dyes. 3 .

3 . The method for preparing a dye for colored polyacrylamide gel according to claim 2 , wherein the molecular structure of the original dye does not contain water-soluble groups. 4 .

4 . The method for preparing a dye for colored polyacrylamide gel according to claim 2 , wherein the carbonyl dye is any one of anthraquinone dyes or thioindigo dyes. 5 .

5. the preparation method of the dye of a kind of colored polyacrylamide gel according to claim 1, is characterized in that, the chemical reaction equation of described gel dye preparation step is as follows:

Among them, under alkaline conditions, sodium dithionate emits electrons, and dye molecules accept electrons and are reduced to soluble sodium salts to obtain gel dye mother liquor that can be used for polyacrylamide gels.

6. A dye for colored polyacrylamide gel, characterized in that, it is prepared by the preparation method according to any one of claims 1-5.

7. the dyestuff of a kind of colored polyacrylamide gel according to claim 6 is characterized in that, the step of applying the dyestuff of described colored polyacrylamide gel to prepare colored polyacrylamide gel is as follows:

S201. Preparation of separating gel: add 30% Acr-Bis, 1.5MT Tris-HCl (pH 8.8), 10% SDS and deionized water according to the concentration of the dyed gel and mix well, then add 10% ammonium persulfate in the formula and TEMED, after mixing evenly, pour the solution between the assembled vertical electrophoresis glass plates quickly. The upper edge of the separating gel is about 3cm away from the edge of the glass plate. The filter paper absorbs the water layer;

S202. Preparation of stacking gel: add 30% Acr-Bis, 1.0M Tris-HCl (pH 6.8), 10% SDS deionized water and an appropriate amount of gel dye mother solution in a beaker according to the proportions and mix them evenly. The final concentration is generally 0.05-1.0 mg/mL, then add 10% ammonium persulfate and TEMED in the formula, mix well, pour the solution into the top of the separating gel until it is full, insert the comb slowly, and let it stand until it solidifies before electrophoresis.

8. an application method of colored polyacrylamide gel, it is characterized in that, the colored polyacrylamide gel obtained according to the described preparation method of claim 7 carries out the separation of protein sample, specifically comprises the following steps:

S301, mixing the stacking gel buffer with gel dye to prepare a colored polyacrylamide gel premix;

S302, when making the gel, mix the separating gel buffer and the separating gel solution uniformly and add the gel to accelerate the gel to obtain direct gel casting;

S303 , when preparing the colored stacking gel, the stacking gel buffer is mixed with the stacking gel solution and a gel accelerator is added to obtain stacking gel gels of different colors, without adding additional dyes.

9. the application of a kind of colored polyacrylamide gel according to claim 8, is characterized in that, described premix is divided into separating gel buffer, separating gel solution, stacking gel buffer, stacking gel solution and gel Coagulants, made from the following ingredients, respectively:

Color stacking gel buffer: 1.0M Tris-HCl (pH6.8), TEMED, dH2O, gel dye;

Separating gel buffer: 1.5M Tris-HCl (pH8.8), TEMED, dH2O;

Separating gel solution: 30% Acr-Bis, dH2O;

Stacking gel solution: 30% Acr-Bis, dH2O;

Gel accelerator: 10% ammonium persulfate.